Technical Field
The present invention relates to Cichorium spp. nuclear recessive male sterile mutants of Cichorium intybus subsp. intybus var. foliosum (also known as leaf chicory), to newly identified polymorphic molecular markers tightly linked to the nuclear recessive gene that control the expression of the male sterile trait in leaf chicory, to methods for the selection of nuclear recessive male sterile mutants in leaf chicory, to methods for the production of inbred lines of leaf chicory showing male sterility (i.e. seed parent) and male fertility (i.e. pollen parent) of leaf chicory, including all cultivated types of Radicchio, and to F1 hybrids that are heterozygous at the locus for male sterility, being so characterized by male fertility.
Description of Related Art
In plant breeding the conventional methods for hybridizing and selecting plants on the basis of the observed phenotype are nowadays not the only methods used by plant breeders. Up to date, molecular genetics and biotechnology are widely used to produce transgenic plants, to produce new mutants and also within breeding programs wherein molecular markers can be essential tools for the selection of the characters of interest. In particular, when new characters of commercial and technical interest are created and/or discovered, a molecular marker for the tracking thereof is extremely useful and can be also the only way for the breeder for early selecting the plants carrying a number of desired characters of interest.
Although part of the steps of breeding programs normally include essentially biological processes such as controlled mating, the resort to technology becomes more and more essential at the initial stages of the breeding programs in order to obtain an early genetic selection of the desired plant phenotypes. In fact, nowadays, crop plants are selected for a high number of desired traits, the selection often being based on pyramiding the superior alleles for several genes. It follows that, at each generation, the genetic recombination through the independent assortment of the genes causes a wide random redistribution of all the alleles including the ones coding for the characters of agronomic and commercial interest. For this reason breeding programs avail themselves more and more frequently of molecular genetics techniques that allow the breeders to carry out a precise selection and/or production of plants expressing the desired characters (corresponding to specific genotypes that combine superior alleles for a number of genes), the techniques being particularly efficient when molecular markers linked to the loci of the genes of interest, said markers being polymorphic and allowing the tracking of the alleles and traits of interest.
Molecular markers with co-dominant inheritance patterns (i.e., capacity to distinguish the heterozygous locus for the two homozygous ones for a given gene or genome sequence), such as microsatellite markers, are of particular interest in order to allow a correct selection of the specific alleles and genotypes desired. A tight linkage of the marker to the locus of interest will result in the co-segregation of a specific allele of the marker with a specific allele of the gene mapping in said locus, thus allowing a very refined tracking of the desired allele/s at that locus. It is therefore essential to identify reliable molecular markers for characters that may be desirable, in particular when the characters of interest are genetically recessive.
In crop plants, commercial F1 hybrids are populations of plants of high commercial interest manifesting extreme vigour, being highly heterozygous (for most of the genes or at least heterozygous for the genes of interest). More precisely, for F1 hybrid plants the term “heterosis” is used, where this term officially indicates in genetics the greater vigour in terms of size, growth rate, resistance to biotic and abiotic stresses, and fertility and productivity of hybrids compared to their parental plants, usually stemmed from controlled crosses between highly inbred lines, which are homozygous for different alleles at each locus being considered. Consequently, heterosis is always associated with increased heterozygosity. These plants are known to produce an F2 progeny (and F3, F4, etc in the following generations) of much lower quality with respect to the F1 generation because of genetic segregation and recombination mechanisms.
The loss of the traits of commercial interest in the generations after F1 is due to the high number of genes of interest for which the F1 plant is heterozygous and to the genetic recombination by means of independent assortment thereof, assortment that will randomly spread the alleles of the genes of interest thus providing F1+n (n≧1) genotypes that are not anymore carrying the desired genotype (and the resulting vigorous phenotype) of F1 in all the loci of interest. It is hence essential, in breeding programs, to be able of tracking the genes of interest, of discriminating between the desired and the undesired alleles thereof, and of generating two antagonist parental lines that are homozygous for different alleles of the same genes of interest that when crossed one with the other will provide, at each hybridization between said parental plants, the desired F1 hybrid. In other words, the crucial part of a breeding program aimed at the constitution of F1 hybrids deals with an accurate genetic selection of the parental plants that is effectively carried out with the aid of molecular genetics techniques.
For the best results in producing F1 hybrids of commercial value, the seed producer parental line (also called “seed parent”) is preferably male sterile thus avoiding completely the occurrence of self-pollination and presence in the F1 generation of inbred progeny seeds in disadvantage to the production of F1 hybrids. For this reason, in absence of an efficient genetic male sterility system, when stamens and pistils occur in the same flower of a fully male fertile seed parent, the plant is normally made male sterile by physical removal of the anthers from the flowers before pollen dispersal.
It is obvious that the introduction or identification of male sterility genes, i.e. genes responsible for the fertility of the male part of the flower that, upon mutation, can provide a male sterile plant would be preferable. Male sterile mutants, that cannot produce viable pollen grains or functional anthers, allow the exploitation of heterosis in F1 hybrid populations of many agricultural and horticultural crops are hence highly desirable.
Two kinds of male sterility can be observed in plants: nuclear and cytoplasmic male sterility. The former type of genetic male sterility is based solely on recessive mutations that affect different functions in nuclear genes (ms indicates the recessive allele causing male sterility whereas Ms indicates the wild type dominant allele rendering the plant male fertile), while cytoplasmic male sterility (CMS) is maternally inherited and mainly due to mutations in the expression of mitochondrial genes that are inherited only maternally by the egg cell cytoplasm. Moreover, in genotypes showing CMS, male fertility can be eventually restored by nuclear-encoded fertility restorer (Rf) genes. In several species, nuclear and/or cytoplasmic male-sterility has been used to produce female parental lines and exploited for the production of hybrid seeds through controlled pollination with male parental lines showing specific combining ability.
Cultivated chicory (Cichorium intybus subsp. intybus L.) is a diploid plant species (2n=18), belonging to the Asteraceae family, subfamily Cichoriodeae, tribe Lactuceae or Cichorieae. These species are naturally allogamous, due to an efficient sporophytic self-incompatibility system. In addition, outcrossing is promoted by a floral morpho-phenology (i.e., proterandry, having the anthers mature before the pistils) unfavourable to selfing in the absence of pollen donors and by a favourable competition of allo-pollen grains and tubes (i.e., pollen genetically diverse from that produced by the seed parents, usually called auto-pollen). Long appreciated as medical plants by ancient Greeks and the Romans, leaf chicory varieties are nowadays amongst the most important cultivated vegetable crops, being used mainly as component for fresh salads or more rarely cooked according to local traditions and alimentary habits. At present, this species are grown all over continental Europe, in South Western Asia, and on limited areas in Northern America, South Africa, and Australia.
Two main groups can be recognized within C. intybus subsp. intybus to which all the cultivated types of chicory belong: the first, which refers to the var. foliosum, traditionally includes all the cultivar groups whose commercial products are the leaves (i.e. leaf chicory), while the second regards the var. sativum and comprises all the types whose commercial product, either destined to industrial transformation or direct human consumption, is the root (i.e. root chicory) (for the taxonomic classification of Cichorium intybus botanical varieties, see Lucchin M., Varotto S., Barcaccia G. and Parrini P. (2008). Chicory and Endive. In: Handbook of Plant Breeding, Vegetables I: Asteraceae, Brassicaceae, Chenopodicaceae. Edited by Jaime Prohens-Tomás and Fernando Nuez. Springer Science, New York, USA. pp. 1-46). The cultivar groups of leaf chicory include mainly Witloof chicory, Pain de sucre, Catalogne and Radicchio. In particular, “Radicchio” is the Italian common name that has been adopted by all the most internationally used languages to indicate a very differentiated group of chicories, with red or variegated leaves, traditionally cultivated in North Eastern Italy. All the red types of Radicchio now being cultivated seem to derive from red-leaved individuals firstly introduced in XV century. According to historical information (Bianchedi A. (1961) I radicchi di Treviso. L'Italia Agricola. 1: 37-51), the cultivation of red chicory goes back to the first half of XVI century. For sure, the original type has to be identified with the “Rosso di Treviso” which has been for long the only cultivated Radicchio in the Venetian territories. Originally selected around 1930, nowadays “Rosso di Chioggia” is by far the most widely grown among the various types of Radicchio and the one which presents the highest within-type differentiation as far as the availability of cultivars able to guarantee an almost complete year round production. As a matter of fact, it has shown a great adaptability to very different environmental situations all around the world, becoming the most grown type of Radicchio outside the Italian country and the most known at international level (Lucchin et al., 2008).
It is worth mentioning that traditionally cultivated populations of leaf chicory, in general, and radicchio, in particular, were developed by mass selection in order to obtain uniform populations characterized by valuable production and acceptable commercial head size and shape. Newly released varieties are mainly synthetics produced by intercrossing a number of phenotypically superior plants, selected on the basis of morpho-phenological and commercial traits. More rarely, plants are also evaluated genotypically by means of progeny tests. Synthetics have a rather large genetic base and are represented by a heterogeneous mixture of highly heterozygous genotypes sharing a common gene pool. In recent years, methods for the constitution of F1 hybrids have been developed by private breeders and seed firms. Details on the procedure for the constitution of such hybrids are not available in the current literature and it may be presumed that each company has developed its own protocol, mainly in accordance to the genetic material it has at disposal and to the possibility of applying a more or less efficient control on the F1 hybrid seed production phase.
As a matter of fact, the strong self-incompatibility system, which hinders obtaining highly homozygous parents, and the absence of a male-sterility factor within the species or in sexually compatible species, made it generally difficult to propose an efficient F1 seed production scheme and, most of all, to consider these newly commercial populations or varieties as true F1 hybrids for leaf chicory.
As it happens for most allogamous species, in leaf chicory detectable heterosis effects are present and hybridization between genotypes selected on the basis of their specific combining ability gives vigorous and uniform progenies. Consequently, the constitution of F1 hybrid populations is profitable in a practical breeding scheme and it is also feasible on a large commercial scale by the selection of self-compatible genotypes, for the production of inbred lines, and the identification of genotypes showing male-sterility, to be used as see parents for the hybridization with pollen donors. It is therefore expected that F1 hybrid populations will be bred and adopted with increasing frequency for leaf chicory. This is particularly true for the cultivated types that take a great advantage from the uniformity of the marketed products, as this is often the key for the customer's appreciation.
Notwithstanding the high commercial interest, the presence of a naturally occurring CMS system has not been reported in leaf chicory whereas strategies to genetically engineering male sterility were used in Magdeburg, Witloof and Chioggia genotypes (reviewed in Lucchin M., Varotto S., Barcaccia G. and Parrini P. (2008). Chicory and Endive. In: Handbook of Plant Breeding, Vegetables I: Asteraceae, Brassicaceae, Chenopodicaceae. Edited by Jaime Prohens-Tomas and Fernando Nuez. Springer Science, New York, USA. pp. 1-46).
In a first approach, transgenic male sterile lines of leaf chicory were produced by expressing the ribonuclease gene RNase from Bacillus amyloliquefaciens (known as BARNASE) under the control of a tapetum-specific promoter originally isolated from tobacco (TA-29) (see Mariani C., De Beuckeleer M., Trueltner J., Leemans J and Goldberg R. B. (1990). Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature, 347: 737-741). Restorer lines for these male-sterile lines were obtained by expressing the gene coding for the so-called BARSTAR, the intracellular inhibitor of BARNASE under control of the same promoter (Denis M., Delourne R., Gourret J. P., Mariani C. and Renerd M. (1993). Expression of engineered nuclear male sterility in Brassica napus: genetics, morphology and sensitivity to temperature. Plant Phys., 101(4): 1295-1304; Reynaerts A., Van de Wiele H., de Sutter G. and Janssens J. (1993). Engineered genes for fertility control and their application in hybrid seed production. Sci. Hort., 55: 125-139). The development of inbred lines and male-sterile lines provided a reliable pollination control and allowed a new hybrid seed production system, which has been registered as SeedLink™. This system for genetically engineering pollination in plants was invented and implemented by the private industry Plant Genetic Systems (Belgium).
Somatic hybridization by means of protoplast symmetric fusion between chicory and the CMS line of sunflower PET-1 was also attempted in order to promote the regeneration of interspecific hybrid plants. This kind of CMS in sunflower was identified in an interspecific cross between Helianthus petiolaris and Helianthus annuus, and it was associated with the expression of the mitochondrial gene ORF522, encoding a 15-kD polypeptide. The ORF522 gene was originated by a recombination event at the 3′ of atp1 gene and its protein is detectable in flowers of CMS but not of restored lines (Horn R., Köhler R. H. and Zetsche K. (1991). A mitochondrial 16-kDA protein is associated with cytoplasmic male sterility in sunflower. Plant Mol. Biol., 17: 29-36; Monegèr F. and Smart C. J. (1994). Nuclear restoration of cytoplasmic male sterility in sunflower is associated with the tissue-specific regulation of a novel mitochondrial gene. EMBO J., 13(1): 8-17). The hybrid plants obtained after somatic symmetric fusion were cytoplasmic hybrids, cybrids, and showed mtDNA rearrangements, indicating that symmetric fusion had the tendency to maintain the chicory mitochondrial genome. Three different kinds of sterility were observed: i) plant with anthers lacking dehiscence without, or with non-viable, pollen; ii) complete absence of the anthers; and iii) absence of both anthers and styles or the presence of reduced styles. One of these male-sterile plants was used for the production of F1 hybrids whose yields were equal to or higher that those of traditional varieties (Rambaud C., Dubois J. and Vasseur J. (1993). Male-sterile chicory cybrids obtained by intergeneric protoplast fusion. Theor. Appl. Genet., 87: 347-352; Rambaud C., Bellamy A. Dubreucq A., Bourquin J-C. and Vasseur J. (1997). Molecular analysis of the fourth progeny of plants derived from cytoplasmic male sterile chicory cybrid. Plant Breed., 116: 481-486).
In a subsequent work, three different CMS chicory cybrids were backcrossed to Witloof chicory in order to transfer the male sterile cytoplasm from an industrial chicory to a Witloof genetic background. The transcript analysis revealed that the ORF522 is weakly expressed or not expressed at all in the cybrids. This finding led Dubreucq et al. (1999) to conclude that ORF522 cannot be associated to the CMS observed in the chicory cybrids and to suggest that they presented a novel form of CMS, different from that of sunflower. Protoplast asymmetric fusion was used to produce male sterile somatic hybrids between a Rosso di Chioggia genotype and a PET-1 sunflower CMS line. At anthesis the regenerated cybrids had fewer and non-viable pollen grains but they could set seeds when free-pollination occurred (Varotto S., Nenz E., Lucchin M. and Parrini P. (2001). Production of asymmetric somatic hybrid plants between Cichorium intybus and Helianthus annuus. Theor. Appl. Genet., 102: 950-956). Overall results collected so far using interspecific protoplast fusion experiments suggest that male-sterile cybrid plants can be actually regenerated in chicory. Nevertheless, it appears that mitochondrial genome re-arrangements lead to the creation of novel CMS chicory types instead of transferring the desired trait from CMS sunflower lines. The methods of transgenesis useful for making cytoplasmic male sterile chicory plants comprising the ORF 522 of Helianthus annuus was patented by Delesalle et al. (2004, see U.S. Pat. No. 6,803,497). As a matter of fact, the development of inbred lines and male-sterile lines based on this biotechnological approach failed to provide any reliable hybrid seed production system in chicory.
No endogenous recessive nuclear gene providing upon mutation a male sterile phenotype has so far been identified in leaf chicory (Cichorium intybus subsp. intybus var. foliosum), while a male sterile mutant having a not well-defined genetic inheritance has been reported for root chicory (Cichorium intybus subsp. intybus var. sativum). The latter mutant, apparently characterized by functional male sterility although not cytologically documented by Desprez et al. (Desprez B. F., Delesalle L., Dhellemmes C. and Desprez M. F. (1994) Génétique et amélioration de la chicorée industrielle. CR Acad. Agr. Fr. 80(7): 47-62) has been patented in the République Française on 1 Feb. 2002 by NUNHEMS ZADEN BV (Stérilité male de legumes de Cichorium cultivé et utilisation pour la production de semences hybrides, see No de publication: FR2832290). Recently, the use of high-density molecular maps allowed the fine mapping of molecular markers linked to the genomic locus involved in nuclear male sterility (termed NMS1): in particular, the gene responsible for male sterility trait in root chicory was found associated to the linkage group 5 of Cichorium intybus L. (Gonthier L., Blassiau C., Mórchen M., Cadalen T., Poiret M., Hendriks T., Quillet M. C. (2013) High-density genetic maps for loci involved in nuclear male sterility (NMS1) and sporophytic self-incompatibility (S-locus) in chicory (Cichorium intybus L., Asteraceae). Theoretical and Applied Genetics, 126(8): 2103-2021. doi: 10.1007/s00122-013-2122-9).
Concerning markers, only a few genetic studies using molecular markers have been carried out on Cichorium spp. mainly to characterize commercial varieties and experimental materials, to evaluate the genetic homogeneity and purity, respectively, of inbreds and hybrids, and to investigate phylogenetic relationships between cultivars and cultivar groups of C. intybus and other species, both cultivated and wild, belonging to the same genus. Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) markers were also used to construct the first genetic map of C. intybus. More recently, a new genetic map was constructed for chicory using simple sequence repeat (SSR or microsatellite) markers by Cadalen et al. (Cadalen T., Mörchen M., Blassiau C., Clabaut A., Scheer I., Hilbert J-L., Hendriks T. and Quillet M-C. (2010). Development of SSR markers and construction of a consensus genetic map for chicory (Cichorium intybus L.). Molecular Breeding, 25: 699-722). This consensus genetic map, which includes 9 homologous linkage groups one for each of the 9 haploid chromosome complements, was obtained after the integration and ordination of molecular marker data of one witloof chicory and two industrial chicory progenies.
It is worth emphasizing that molecular markers in Cichorium spp. have been exploited for selecting the mother plants of synthetics as well as for determining the distinctiveness, uniformity and stability, i.e. DUS testing, of newly bred varieties. In Cichorium spp., molecular markers should also find utility for assessing the genetic homogeneity and homozygosity of inbred lines produced by repeated selfing, measuring the genetic diversity among inbred lines in order to plan crosses and maximize heterosis in the experimental F1 hybrids, and evaluating the genetic purity and heterozygosity of seed stocks of commercial F1 hybrids.
In conclusion, providing male-sterility in the leaf chicory species will open new frontiers for breeding new varieties in general, especially if this trait can be profitably transferred to elite lines and precociously identified by molecular diagnostic assays suitable to perform marker-assisted selection programs.